Introduction
Type II diabetes mellitus is a rapidly growing worldwide health challenge. Diabetic cardiomyopathy is a heart muscle disease characterised by progressive contractile dysfunction independent of coronary artery disease or hypertension. It is driven by metabolic dysfunction, oxidative stress, calcium mishandling and mitochondrial impairment. Classical disease models are limited in their ability to replicate human-specific disease mechanisms. Therefore, we used induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) to provide a strong patient-specific model for investigating cellular and molecular changes in diabetic cardiomyopathy, as they retain donor-specific metabolic and epigenetic characteristics.
Methods
iPSCs generated from three diabetic and three non-diabetic donors were differentiated into cardiomyocytes through temporal modulation of Wnt/β-catenin signalling. The iPSC-derived cardiac cultures contained a heterogeneous population of cardiomyocytes (CMs) and cardiac fibroblasts (CFs), better recapitulating the native myocardial microenvironment. Molecular and functional comparisons were performed using immunofluorescence, RT-qPCR, Western blotting, RNA sequencing, and live-cell fluorescence assays. Functional phenotyping included measurements of mitochondrial reactive oxygen species (ROS), apoptosis, and Ca²⁺ dynamics.
Results
Diabetic iPSC-CMs exhibited increased oxidative stress, elevated apoptosis, and a marked increase in hypertrophic markers associated with cardiac stress, including ANP, BNP, GATA4, and ANKRD1, as well as fibrotic markers including TGFB1, CTGF, FN1, POSTN, COL1A1, and COL3A1. Flow cytometry revealed increased forward scatter (FSC) in diabetic TNNT2+, confirming a hypertrophic phenotype. Furthermore, higher FSC-A of vimentin+ and PDGFRα+ populations, along with a higher proportion of α-SMA-positive cells, indicated increased cardiac fibroblast proliferation and myofibroblast activation. Calcium imaging revealed profound defects during excitation–contraction coupling, characterised by elevated baseline cytosolic Ca²⁺, reduced sarcoplasmic reticulum Ca²⁺ stores, and delayed Ca²⁺ reuptake, consistent with impaired diastolic relaxation.
RNA sequencing of iPSC-CMs from three donors per group identified 1,412 differentially expressed genes. Pathway enrichment and gene ontology analyses revealed significant upregulation of gene networks related to cardiac hypertrophy, fibrosis, ECM remodelling, and ion channel regulation in diabetic iPS-CMs. The WNT/β-catenin and TGF-β signalling pathways were prominently enriched, both of which serve as key drivers of maladaptive hypertrophic and fibrotic remodelling in the diabetic cardiac phenotype.
Pharmacological inhibition of Wnt signalling partially rescued the diabetic phenotype, reducing oxidative stress and improving calcium handling in contractile cardiomyocytes. attenuating TGFβ signalling, as demonstrated by reduced SMAD2/3 activation and decreased secretion of TGFβ1 and PAI-1, in addition to hypertrophic markers such as secreted ANP.
Conclusion
Collectively, this study shows that donor-specific iPSC-CMs can recapitulate key features of diabetic cardiomyopathy and provides mechanistic insights, linking aberrant signalling pathways to functional impairment. This work highlights the potential of testing donor-specific therapeutic targets and establishes a clinically relevant platform for treating diabetes-associated heart disease.